Universal variable fragments

ABSTRACT

Methods and kits for analyzing a subject&#39;s genomic DNA to determine the subject&#39;s lineage. The methods comprise providing oligonucleotide primers for use in nucleic acid amplifications on a subject&#39;s genomic DNA. A first oligonucleotide primer includes a repeat sequence and at least one non-repeat nucleotide located on its 5′ end. A second oligonucleotide primer starts within an amplification-permissive genetic distance on the 3′ side of the repeat sequence in the subject&#39;s genomic DNA and may include an a-selective base, such as inosine. Additional oligonucleotide primers may be provided. The methods further comprise conducting nucleic acid amplifications on the subject&#39;s genomic DNA using the oligonucleotide primers of the invention to produce amplified DNA fragments based on repeat sequences found at the 5′ end of the subject&#39;s genomic DNA and analyzing such amplified DNA fragments to determine the length of repeat sequences found in the subject&#39;s genomic DNA.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/958,221, filed Apr. 15, 2002, now U.S. Pat. No. ______, issued,______, 2003, which is a national stage of international applicationPCT/NL01/00177, filed Mar. 5, 2001.

TECHNICAL FIELD

The invention relates generally to methods and materials for the geneticanalysis of a subject.

BACKGROUND

For some species, reliable, simple technologies are available forgenetic analysis of individuals. However, for most animal and birdspecies, genetic information is insufficient for applied genetics.Developing existing technologies for each species to obtain genetic datawill be extremely laborious and time consuming. Progress to date hasbeen slow. The situation is particularly problematic in the area ofwildlife management. For example, building DNA patterns of hawks iscurrently almost impossible. At the same time, there have been reportsof people illegally placing eggs from wild mating hawk couples in tamedbreeding hawk nests. It is currently nearly impossible to prove fraudusing DNA data in species where genetic variation has not beenpreviously described.

Other areas of interest are DNA identification of exotic species (e.g.,animals, plants, organisms) for various reasons. For instance, animalsarriving through veterinary control can be identified by sampling themboth at departure and at arrival. Using the animal's individual DNA toidentify it, animals can be tracked, and proof of their origin is alwayspossible.

Furthermore, parentage verification in rare, expensive animals andstrain identification of plants can be performed for any givencombination or species. Reports have been made of selling the offspringof “lower” breeding parents as the highest possible quality been made ofselling the offspring of “lower” breeding parents as the highestpossible quality animals.

Another problem is the determination of sex. For many exotic species,genetic markers are not available to perform sex determination.

A need exists for a method of quickly genetically analyzing a species todetermine, among other things, its lineage, sex, and origin.

BRIEF SUMMARY OF THE INVENTION

The invention provides a new technology which has been developed for thequick genetic analysis of a species and individuals thereof. The methodincludes the use of first and second oligonucleotide primers forperformance of PCR amplification on the genomic DNA. The firstoligonucleotide primer is a 5′ variation generator, including a repeatsequence and at least one non-repeat nucleotide. The secondoligonucleotide primer is a 3′ fragment generator starting within such agenetic distance that amplification of the genomic DNA can be performedand preferably includes inosine. PCR amplification of the genomic DNA isconducted at a relatively low annealing temperature using both the firstand second oligonucleotide primers under conditions such thatessentially neither the first nor the second oligonucleotide primeralone can amplify sufficient DNA to be detected. DNA fragments are thusproduced based on repeat sequences on one end of the genomic DNA, andother sequences based on the opposite end of the genomic DNA. Theresulting PCR products can then be analyzed for the length of a repeatsequence found in the genome. A second PCR is preferably conducted onthe diluted PCR products of the first PCR. Such a second PCR would beconducted using third and fourth oligonucleotide primers. The third andfourth oligonucleotide primers are elongated versions of the first andsecond oligonucleotide primers, respectively, thus enabling PCRamplification at relatively higher annealing temperatures and enabling aselection of a sub-set of the DNA fragments amplified in the first PCR.At any point, an optional but preferred restriction digestion may beconducted. The technology has been developed for the quick geneticanalysis of a species which is reliable, reproducible, simple, anduseful for all species/organisms (e.g., animal, avian, bacterial, viral,and plant). The invention particularly relates to samples obtainablefrom non-human species but is applicable to samples obtained from humansas well. Neither variation in genome length nor genome compositionappears to influence or limit the characteristics of the technology.This new technology is generally reliable, reproducible, simple, anduseful for all species/organisms (e.g., animal, avian, bacterial, viral,and plant). Furthermore, any material containing DNA (e.g., blood, hairfollicles, etc.) can be used as a source for the generation of DNApatterns.

In one aspect, the invention includes a method of analyzing genomic DNAin a sample. This method includes providing first and secondoligonucleotide primers, wherein the first oligonucleotide primer is a“5′ variation generator” comprising a repeat sequence and at least onenon-repeat nucleotide on the first oligonucleotide's 5′ end. Meanwhile,the second oligonucleotide primer is a “3′ fragment generator” startingwithin such a genetic distance that amplification of the genomic DNA canbe performed. A nucleic acid amplification such as a polymerase chainreaction (“PCR”) amplification is conducted on the genomic DNA in thesample using both the first and second oligonucleotide primers. Thenucleic acid amplification is conducted under conditions such thatneither the first nor the second oligonucleotide primer alone amplifiesDNA, thus producing DNA fragments based on repeat sequences on one endof the genomic DNA and other sequences based on the opposite end of thegenomic DNA. The amplified products are then analyzed to determine thelength of a repeat sequence found in the genomic DNA, which can becompared with the DNA putatively of the same individual or the DNA ofthe individual's putative ancestors or relatives.

Alternatively, and as more thoroughly described hereinafter, multipleamplifications and/or restriction digestion might also be used with thetechnique.

As described herein, the first primer, the “5′ variation generator,”includes a complementary repeat sequence and at least one non-repeatnucleotide so as to start the amplification at a repeat sequence of thegenomic DNA.

By localizing the 5′ variation generator at the 5′ site of repeatsequences, the repeat length variation is enclosed in the amplificationrounds. Primers are thus bound to hybridize at the 5′ ends of repeatsequences by addition of one or more nucleotides at the end of theprimer.

While the oligonucleotide primers at repeat sequences provide detectionof genetic variation, the 3′ fragment generator is used to amplifyfragments of reasonable sizes (e.g., 100 base pairs to 10,000 basepairs). The 3′ fragment generator starts within such a genetic distancethat amplification of a sample DNA can be performed and preferablyincludes inosine or another a-selective base allowing it to influenceannealing temperatures without coincident or equal influence on thestringency of the annealing reaction. The 3′ fragment generator isdesigned to anneal to the DNA within a short distance, as mentionedbefore. To do this, the number of selective nucleotides is kept at a lownumber, whereas the annealing temperature is influenced by a number ofnon-selective nucleotides, such as inosines, universal bases, and anycombination of A, C, G or T (e.g., R, Y, N). By using such a 3′ fragmentgenerator, the invention provides optimal reaction conditions in thereaction that are generally well suited to the reaction conditionsrequired for the 5′ generator. In short, the number of selectivenucleotides of this primer is maintained at a relatively low number,whereby the annealing temperature is raised to enable reliable andreproducible amplification using a-selective bases, such as inosines, inthe fragment generator oligonucleotide.

Some of the genetic markers identified using the technology will belocated on the male and female sex chromosomes. After the identificationof such markers, these markers can be used to determine the sex ofspecies which are difficult to establish through phenotypiccharacteristics (e.g., porcupine or crocodile).

The invention also includes a kit of parts for performing the geneticanalysis and a method of manufacturing such kit for use in geneticanalysis. The invention is further described in the detailed descriptionwithout limiting the invention thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The samples used in the illustrations are based on high molecular weightDNA obtained from blood samples from each animal.

FIG. 1 illustrates the analyses of five species. Clear differences arepresent. Different lanes present 1) horse, 2) parrot, 3) cattle, 4)ostrich and 5) pig. The illustration shows DNA fragments ranging fromsizes between 100 and 1200 bp.

FIG. 2 is an illustration of the analyses of five species. Cleardifferences are present. Different lanes present 1) horse, 2) parrot, 3)cattle, 4) ostrich and 5) pig. The illustration shows DNA fragmentsranging from sizes between 250 and 300 bp.

FIG. 3 depicts the variation within species. Two samples of the samespecies (ostrich) are presented. At least three loci are presented.

DETAILED DESCRIPTION OF THE INVENTION

To combine the amplification of many DNA fragments with the selection ofa specific set of informative DNA fragments, a preferred protocol isused which is based on two subsequent PCR amplifications. PCR is one ofmany well-known amplification methods known in the art and is,therefore, not described further here.

In a preferred method, genomic DNA from the sample is amplified in afirst PCR at relatively low annealing temperatures. The 5′ variationgenerator and the 3′ fragment generator are used to generate fragmentsof which a selected part is to be used in a second PCR. The first PCR isusually run under conditions under which neither the 5′ variationgenerator nor the 3′ fragment generator alone amplify DNA. Thus, whenDNA amplification is performed using both the 5′ variation generator andthe 3′ fragment generator, many resulting fragments are based on repeatsequences on one end of the genomic DNA and, at the same time, manysequences are based on an opposite end of the genomic DNA.

After possible dilution of the PCR products of the first PCR, a secondPCR is preferably performed. This second PCR is conducted using thirdand fourth oligonucleotide primers. The third and fourth oligonucleotideprimers are commonly elongated versions of the first and secondoligonucleotide primers, respectively, thus enabling PCR amplificationat relatively higher annealing temperatures and enabling a selection ofa sub-set of the DNA fragments amplified in the first PCR. The fourtholigonucleotide primer preferably includes inosine residues.

At any point during this procedure, a preferred, but optional,restriction digestion may take place. Another source of geneticvariation in amplified fragments is the presence or absence ofrestriction sites. Addition of a restriction digest after the second PCRincreases the number of genetic polymorphisms detected. Furthermore, thesizes of the DNA fragments to be analyzed for their length are decreasedas well.

The amplified PCR products can then be analyzed using a variety ofexisting methods.

As can be determined, many DNA fragments are amplified in the first PCRamplification, whereas a subset of these DNA fragments are multiplied inthe second PCR amplification. Both reactions are preferably run understringent conditions. Primers used in the PCR procedure can vary inlength. Lengths between 4 and 50 nucleotides or inosines were used inthe examples.

Primer Design

As previously identified, the variation generator starts at a repeatsequence, while the fragment generator starts within such a geneticdistance that amplification of the DNA can be performed.

For the 5′ variation generator, repeat sequences exist throughout anygenome in many variations, such as mononucleotide (A, G, C or T) repeat,dinucleotide (CT, CA, CG, AT, AC, AG, GT, GC, GA, TA, TG and TC) repeat,trinucleotide (e.g., TGA, CTG, etc.) repeat, tetranucleotide (e.g.,TGCA, CTGT) repeat, and so forth. For instance, an AC repeat can havethe structure: CACACACACACA (SEQ ID NO:1) (“6-repeat”), or CACACA (SEQID NO:2) (“3-repeat”).

Repeat sequences, of course, also exist in the as yet unanalyzed genomesof species. Repeat sequences exhibit different lengths due to the numberof repeats present. For different individuals, differences exist in thenumbers of repeats in each locus (“microsatellite”). Thus, geneticvariation in repeat sequences can be determined based upon lengthvariation caused by the number of nucleotide repeats in a locus. Thenumber of repeats in a microsatellite can vary enormously with differentindividuals of the species. Many sequences contain a few repeats (e.g.,2 or 3), whereas some repeats are known to include thousands of basepairs (“bp”).

By localizing the oligonucleotide primer at the 5′ site of repeatsequences, as described herein, the repeat length variation is enclosedin the amplification rounds which are part of PCR. Primers are forced tohybridize at 5′ ends of repeat sequences by adding one or morenucleotides which do not continue the repeat pattern at the 5′ end ofthe primer. This result is due to the nature of the amplifying enzyme,which elongates DNA fragments starting from the 3′ end ofoligonucleotide primers.

While the choice of oligonucleotide primers at repeat sequences providesmost of the detection of genetic variation, the 3′ fragment generator isessentially used to amplify fragments of reasonable sizes (100 bp to10,000 bp).

The number of selective oligonucleotides of the primer is maintained ata low number. At the same time, the annealing temperature is raised toenable reliable and reproducible amplification. This is done usinginosine substitutions in the fragment generator. Inosines are used toincrease annealing temperatures without affecting the binding conditionsof oligonucleotides. Inosines match to any of the four nucleotides inthe DNA. When inosine is substituted for a nucleic acid, it contributesto the sensitivity of the technique.

PCR

To combine the amplification of many DNA fragments with the selection ofa specific set of informative DNA fragments, a protocol is used which isbased on two subsequent PCR amplifications. In the first PCRamplification, many DNA fragments are amplified, whereas in the secondPCR amplification, a subset of these DNA fragments are multiplied. Bothreactions are run under stringent conditions.

After the PCR (or PCRs) have been conducted, the resulting amplifiedfragments are preferably subjected to restriction digestion to determinethe presence or absence of restriction sites. Use of the restrictionenzymes increases the number of genetic polymorphisms detected.

The (preferably digested) product is then sequenced using techniquesknown in the art to determine the particular genetic patterns or markerspresent, when so desired.

Applications

The nature of universal variable fragments (UVF) combines flexibilityand reproducibility with high levels of polymorphisms. The 5′ variationgenerator (based on the microsatellite sequence) mostly corresponds withthe genetic variation typically found in microsatellites, whereas the 3′fragment generator is mainly linked to presence/absence polymorphisms.This strategy is typically based on the use of two different fluorescentlabels-one associated with the 5′ variation generator, the othercorresponding to the 3′ fragment generator. This concept enables theoptimal use of high throughput analysis systems based on multiplefluorescent dyes.

In comparison with other technologies, e.g. AFLP, UVF has an increasedpower to generate polymorphisms in search for high marker density. Onedistinct advantage of the UVF system is found in the possibility toincrease the marker density in regions of chromosomes of specificinterest by choosing the order of the bases of the 3′ fragmentgenerator, instead of random, in the flanking region of a known geneticmarker. As a result, a number of genetic markers can be identifiedwithin a short distance from, e.g., QTL markers. This prospect is notpossible with other technologies such as AFLP (and SAMPL), or ISSR(Inter Simple Sequence Repeat).

Compared to several technologies, UVF is different:

a) The power to generate polymorphisms is much larger compared to RAPD(random amplified polymorphism detection). Due to its concept of athree-step strategy, UVF has increased power to generate polymorphisms.RAPD is based on only one primer in just one PCR, whereas UVF istypically based on two consequent PCR amplifications, followed by adigestion step.

b) Amplification using ISSR (Inter Simple Sequence Repeat) is based onone primer in one PCR reaction. UVF is completely different based ontypically two PCR amplifications and the use of a digestion step.

c) Amplified Fragment Length Polymorphisms (AFLP) is based on the use ofadaptor ligation to initiate PCR. This procedure is typically completelyabsent in UVF, as is the obligation to start the reaction with adigestion of several restriction enzymes.

d) SAMPL is completely based on AFLP, but is directed to the detectionof microsatellites using the AFLP technology.

Furthermore, compared to RAPD and microsatellite analysis, the power ofthe present invention to generate large amounts of polymorphisms from asmall amount of genomic DNA is clear.

Several areas for applications based on UVF include:

1. Gene Hunting

The detection and identification of genetic markers for diseases orbeneficial genetic characteristics is possible using UVF. Due to itseffectiveness, even in species with a relatively well-developed geneticmap, UVF is useful. In other species where the number of availablegenetic markers is low, UVF will be the technology of choice.

2. Marker Density

In situations where QTL analysis has revealed a genetic marker (e.g.,RFLP or microsatellite) with known sequence, the number of markers in adefined region can be increased using the UVF technology. This can beachieved by locating the nine bases of the 3′ fragment generator in theflanking region of the polymorphic marker. This enables generation ofgenetic markers in specific areas of interest. Using this approach, therange in which QTLs may be located can be decreased, and, for example,“candidate gene approach” can be more directed.

This strategy can be further used to detect genetic variation in thegenomic regions close to promoter sites located close to genes. Thedesign of the 3′ fragment generator can be based on general promotersites or on sequences recognized by transcription factors. This furtherillustrates the power of UVF over other available technologies.

3. Forensic Analysis

Due to the nature of UVF, small quantities of DNA can be used togenerate DNA profiles. This enables the use of UVF in situations whereonly limited amounts of DNA are available for genetic analysis, e.g.,forensics.

4. Biodiversity

UVF enables the search for breed- and species-specific markers. Amongother issues, (sub)-species identification of, for example, birds willsolve many enduring discussions.

In use, the invention is quite straightforward. The invention providesrapid and straightforward identification of endangered animals andplants. Many wildlife species, both animals and plants, are protected bylaw. Only limited numbers of individuals may be kept in private.However, identification and lineage of these individuals needs to beproven to effectively protect the law. The invention provides the meansto answer any question in wildlife management relating to identity orlineage, also of species of which specific sequences are little known.The invention also provides genetic maps of a species. In some species,genetic information, and certainly genetic maps, are underdeveloped.Usually, identification of genetic markers is time and labor consumingusing the existing methodologies (e.g. microsatellites).

Furthermore, testing of these markers is inefficient due to the lownumber of genetic markers amplified in one, single reaction.

With this new technology, genetic markers are developed at low cost withhigh speed and efficiency. Thus, “classical” laborious methods are nolonger needed and no individual primer sets for each marker is needed.Furthermore, using the invention, many genetic markers can be identifiedand analyzed in a short period of time. Further analysis of thesegregation of these markers in families where diseases, resistancegenes, or other genes of interest are segregating as well will enablethe identification of genetic markers related to the genes of interest.

In the case where lineage is in question, once the genetic markers ofthe individual have been determined, they can likewise be determined forthe putative parents. The sets of markers (e.g., the number of repeatsin a locus, the length variation of the set of amplified fragments, andso on) from an individual can then be compared with the markers of theputative parent or parents, and a determination of lineage made. In thecase of the aforementioned hawk, for instance, if the number of repeatsin a particular locus of the hawks' DNA do not match that of the tamedbreeding putative parents, the conclusion can be drawn that the tamedbreeding pair are not the parents.

The same situation arises when the question of pedigree arises. Thegenetic markers of the individual are compared and contrasted with thoseof the putative ancestors or relatives, especially parents.

In the case of gender determination, a library would first beconstructed of the particular species (e.g., crocodile) with particularemphasis put on the Y chromosome. Once conserved genetic markers presenton the Y chromosome are identified for the species, the DNA of theindividual in question can be analyzed with the instant invention.

When the question centers around whether or not an individual of aspecies is the same individual previously tested (e.g., by a nation'shealth, agricultural, or racing authorities), the individual is testedat a different time, and the results are compared with those of theearlier analysis.

A kit of parts for use with the invention includes first and secondoligonucleotide primers for performance of the first polymerase chainreaction amplification on the genomic DNA of the individual. The firstoligonucleotide primer is a 5′ variation generator, starting at a repeatsequence. The second oligonucleotide primer is a 3′ fragment generatorstarting within such a genetic distance that amplification of thegenomic DNA can be performed. The kit will also preferably includecomponents for performing the second PCR. Such components include thirdand fourth oligonucleotide primers, wherein the third oligonucleotideprimers is an elongated version of the 5′ variation generator, and thefourth oligonucleotide primers is an elongated version of the 3′fragment generator. The kit of parts further also preferably includesappropriate restriction enzymes.

The invention is further explained by use of the following illustrativeexamples. In the examples, only a limited number of primers are shown.However, any combination of primers based on the information presentedherein is considered to be using the same principles of this technology.

EXAMPLES Example I

Primer Design

Using the previously described criteria (e.g., starting at a repeatsequence and localizing the variation generator oligonucleotide primerat the 5′ site of repeat sequences, and starting within a geneticdistance), the hereinafter described examples of feasible primersequences were determined. As can be seen, the 5′ (left) end of thevariation generator includes a nucleotide which is not consistent withthe existing repeat pattern of the repeat sequence.

PCR 1. Variation Generator. TTGTGTGTG (SEQ ID NO:3) ATGTGTGTG (SEQ IDNO:4) CTGTGTGTG (SEQ ID NO:5) CCACACACA (SEQ ID NO:6) GCACACACA (SEQ IDNO:7) TCACACACA (SEQ ID NO:8) TTTGTGTGTG (SEQ ID NO:9) ATTGTGTGTG (SEQID NO:10)

Fragment Generator. ATGTIIIIIT (SEQ ID NO:11) ATGTCIIIIT (SEQ ID NO:12)ATGTCTIIIT (SEQ ID NO:13) TIIITGTCAG (SEQ ID NO:14) TLIIACGTCG (SEQ IDNO:15)PCR 1. Amplification of Many Fragments

Genomic DNA taken from a sample was amplified in a first PCR at lowannealing temperatures (for example, 30-65° C., but preferably lowerthan temperatures used at an optional second round of amplification).The previously described oligonucleotide primers were used to generatefragments. The first PCR was run under conditions where neither thefragment generator primer nor the variation generator primer alone couldamplify DNA.

PCR 2. Amplification of a Subset of PCR 1.

After dilution of the PCR products of the first PCR, a second PCR wasconducted using the resulting fragments. This second PCR was based onthe hereinafter described fragment and variation generators which, ascan be seen, were elongated to enable PCR amplification at higherannealing temperatures (40-70° C.). This enabled the selection of asubset of the DNA fragments amplified in PCR 1.

Examples of the elongated primer sequences:

PCR 2. Variation Generator. TTGTGTGTGTGTGTGTG (SEQ ID NO:16)ATGTGTGTGTGTGTGTG (SEQ ID NO:17) CTGTGTGTGTGTGTGTG (SEQ ID NO:18)CCACACACACACACACA (SEQ ID NO:19) GCACACACACACACACA (SEQ ID NO:20)TCACACACACACACACA (SEQ ID NO:21)

Fragment Generator. Six Examples are Shown: ATGTIIIIITIIIIT (SEQ IDNO:22) ATGTCIIIITIIIITA (SEQ ID NO:23) TIIITGTCAGLIIA (SEQ ID NO:24)TIIITGTCAGIIIAA (SEQ ID NO:25) TIIIACGTCGIIIA (SEQ ID NO:26)TIIIACGTCGIIIAA (SEQ ID NO:27)

The thus amplified PCR products were digested with restriction enzymessuch as BamHI or HinfI increasing the number of genetic polymorphismsdetected and reducing the sizes of the DNA fragments to be analyzed.

Analysis

Analysis of the digested product was conducted on an ABI 377 sequencer(Perkin Elmer, Calif., USA). The detection on this sequencer was madepossible through the use of fluorescently labeled primers; both the 5′variation generator and the 3′ fragment generator were labeled withdifferent dyes (such as FAM, HEX).

Analysis of the various fragment sizes was performed using the softwareGENESCAN™ and GENOTYPER™ (both from Perkin Elmer, Calif., USA).

1-5. (Canceled)
 6. A method of determining the lineage of an individual by analyzing genomic DNA in a biological sample of the individual, said method comprising: analyzing said genomic DNA in said biological sample to determine the presence of a repeat sequence; determining the repeat sequence's length in number of nucleic acids; and comparing the repeat sequence's length with a corresponding repeat sequence length of a putative ancestor of said individual.
 7. The method according to claim 6 wherein the analysis of said genomic DNA in said sample comprises using a first oligonucleotide primer for performing a first amplification on said genomic DNA, said first oligonucleotide primer being a 5′ variation generator and comprising a repeat sequence and at least one non-repeat nucleotide on the first oligonucleotide's 5′ end.
 8. A kit of parts for analyzing genomic DNA in a sample, said kit of parts comprising: first and second oligonucleotide primers for performance of a first nucleic acid amplification on said genomic DNA, said first oligonucleotide primer being a 5′ variation generator, and comprising a repeat sequence and at least one non-repeat nucleotide on the first oligonucleotide's 5′ end, and said second oligonucleotide primer being a 3′ fragment generator.
 9. The kit of parts of claim 8 further comprising: third and fourth oligonucleotide primers, said third oligonucleotide primer comprising the oligonucleotide sequence of said first oligonucleotide primer together with further nucleotides, and said fourth oligonucleotide primer comprising the oligonucleotide sequence of said second oligonucleotide primers together with further nucleotides.
 10. The kit of parts of claim 8 further comprising at least one restriction enzyme. 11-13. (Canceled)
 14. A method for determining the lineage of a subject, comprising: acquiring a sample of the subject's genomic DNA including at least one genomic repeat sequence; identifying the at least one genomic repeat sequence; determining a length of the at least one genomic repeat sequence; and comparing the length of the at least one genomic repeat sequence with a length of a corresponding repeat sequence of a putative relative of the subject.
 15. The method according to claim 14, wherein identifying the at least one genomic repeat sequence comprises: providing a first oligonucleotide primer and a second oligonucleotide primer; and conducting a first nucleic acid amplification on the subject's genomic DNA using the first oligonucleotide primer and the second oligonucleotide primer to produce amplified DNA fragments based on repeat sequences on at least one end of the subject's genomic DNA.
 16. The method according to claim 15, wherein providing the first oligonucleotide primer comprises providing an oligonucleotide primer including a repeat sequence and a non-repeat nucleotide located on the 5′ end thereof, the non-repeat nucleotide serving to localize the first oligonucleotide primer to the 5′ end of the at least one genomic repeat sequence.
 17. The method according to claim 15, wherein providing the second oligonucleotide primer comprises providing an oligonucleotide primer that starts within an amplification-permissive genetic distance on the 3′ side of the at least one genomic repeat sequence.
 18. The method according to claim 15, wherein providing the second oligonucleotide primer comprises providing an oligonucleotide primer including at least one non-selective base.
 19. The method according to claim 15, wherein providing the second oligonucleotide primer comprises providing an oligonucleotide primer including at least one inosine residue.
 20. The method according to claim 15, further comprising: providing a third oligonucleotide primer and a fourth oligonucleotide primer; and conducting a second nucleic acid amplification using the third oligonucleotide primer and the fourth oligonucleotide primer on amplified DNA fragments produced by the first nucleic acid amplification to enable a selection of a sub-set of the amplified DNA fragments produced by the first nucleic acid amplification, wherein the third oligonucleotide primer is an elongated version of the first oligonucleotide primer and the fourth oligonucleotide primer is an elongated version of the second oligonucleotide primer.
 21. The method according to claim 20, wherein providing the fourth oligonucleotide primer comprises providing an oligonucleotide primer including at least one non-selective base.
 22. The method according to claim 20, wherein providing the fourth oligonucleotide primer comprises providing an oligonucleotide primer including at least one inosine residue.
 23. The method according to claim 15, further comprising digesting the products of the first nucleic acid amplification with a restriction enzyme to increase the number of genetic polymorphisms detectable in the subject's genomic DNA and to decrease the sizes of the amplified DNA fragments.
 24. The method according to claim 20, further comprising digesting the products of the second nucleic acid amplification with a restriction enzyme to increase the number of genetic polymorphisms detectable in the subject's genomic DNA and to decrease the sizes of the products of the second nucleic acid amplification.
 25. The method according to claim 15, wherein conducting the first nucleic acid amplification comprises conducting a polymerase chain reaction amplification.
 26. The method according to claim 20, wherein conducting the second nucleic acid amplification comprises conducting a polymerase chain reaction amplification.
 27. The method according to claim 15, wherein conducting the first nucleic acid amplification comprises conducting the first nucleic acid amplification under stringent conditions.
 28. The method according to claim 20, wherein conducting the second nucleic acid amplification comprises conducting the second nucleic acid amplification under stringent conditions.
 29. The method according to claim 20, further comprising diluting the products of the first nucleic acid amplification before conducting the second nucleic acid amplification.
 30. The method according to claim 15, wherein providing the first oligonucleotide primer comprises providing an oligonucleotide primer selected from the group consisting of SEQ ID NOS: 3-10.
 31. The method according to claim 15, wherein providing the second oligonucleotide primer comprises providing an oligonucleotide primer selected from the group consisting of SEQ ID NOS: 11-15.
 32. The method according to claim 20, wherein providing the third oligonucleotide primer comprises providing an oligonucleotide primer selected from the group consisting of SEQ ID NOS: 16-21.
 33. The method according to claim 20, wherein providing the fourth oligonucleotide primer comprises providing an oligonucleotide primer selected from the group consisting of SEQ ID NOS: 22-27.
 34. The method according to claim 14, wherein the putative relative of the subject is a putative parent of the subject.
 35. A kit of parts for conducting a first nucleic acid amplification on a subject's genomic DNA that includes at least one genomic repeat sequence, comprising a first oligonucleotide primer including a non-repeat nucleotide located on the 5′ end thereof and a repeat sequence, the non-repeat nucleotide serving to localize the first oligonucleotide primer to a 5′ end of the at least one genomic repeat sequence; and a second oligonucleotide primer that starts within an amplification-permissive genetic distance on the 3′ side of the genomic repeat sequence.
 36. The kit of parts of claim 35, further comprising the following parts for conducting a second nucleic acid amplification on the products of the first nucleic acid amplification: a third oligonucleotide primer comprising the sequence of the first oligonucleotide primer together with additional nucleotides; and a fourth oligonucleotide primer comprising the sequence of the second oligonucleotide primer together with additional nucleotides.
 37. The kit of parts of claim 35, wherein the second oligonucleotide primer further comprises at least one non-selective base.
 38. The kit of parts of claim 35, wherein the second oligonucleotide primer further comprises at least one inosine residue.
 39. The kit of parts of claim 35, further comprising at least one restriction enzyme.
 40. The kit of parts of claim 36, further comprising at least one restriction enzyme.
 41. The kit of parts of claim 36, wherein at least one of the additional nucleotides of the fourth oligonucleotide primer is a non-selective base.
 42. The kit of parts of claim 36, wherein at least one of the additional nucleotides of the fourth oligonucleotide primer is an inosine residue.
 43. The kit of parts of claim 35, wherein the first oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 3-10.
 44. The kit of parts of claim 35, wherein the second oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 11-15.
 45. The kit of parts of claim 36, wherein the third oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 16-21.
 46. The kit of parts of claim 36, wherein the fourth oligonucleotide primer is selected from the group consisting of SEQ ID NOS: 22-27. 